Glutathione improves low temperature stress tolerance in pusa sheetal cultivar of Solanum lycopersicum

To investigate the impact of Glutathione (GSH) in mitigating low-temperature stress in Pusa Sheetal cv. of Solanum lycopersicum and imparting low-temperature tolerance by evaluating the different physiological responses. The plant under research was also being studied for its growth and stress tolerance. Low temperatures (LT) stress was applied to seedlings with or without GSH application 12 h before LT stress (prophylactic dose), after 12 h-LT (preemptive dose), and post 12-h recovery (curative dose). Different concentrations of GSH [0, G1 (0.5 mM), G2 (1 mM) and G3 (2 mM)] against LT stress were used. Antioxidant activities, photosynthesis, growth, and stress tolerance indices were quantified. LT stress caused an oxidative burst in S. lycopersicum seedlings of the Pusa Sheetal cv. as indicated by increased peroxidation of lipids and H2O2 concentration. Glutathione reductase (GR), superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX) activities were enhanced. The best concentration was G2 (1 mM), which resulted in a rise in antioxidant activity. Moreover, a decline in lipid peroxidation and H2O2 levels was also seen. The purpose of this study is to identify the role of GSH in reducing LT stress and to find the best dose concentration. This is the first report to assess the GSH-mediated LT stress tolerance in S. lycopersicum (Pusa Sheetal cv.). Therefore, exogenous GSH application of optimal concentration of GSH to LT stressed S. lycopersicum can be an effective approach for augmenting the plant detoxification system and promoting its growth and development.


Effect of low temperature (LT) and exogenous GSH concentrations on pusa sheetal cv.
Growth characteristics. LT plants showed a significant decline in nearly all the growth biomarkers involving root and shoot length, overall fresh and dry mass of plant in comparison to control as well as treated plants (Fig. 1). As compared to control, the root length of LT plants was decreased by 2.4 fold while G1 and G3 showed a 1.06 fold and 1.21 fold decrease in root length. On the contrary, G2 plants showed a substantial increase by 0.9 fold as compared to control plants. G1C, G2C and G3C plants displayed 1.7 fold, 1.5 fold and twofold decrease as compared to control plants, respectively. In the case of shoot length LT and G3 plants showed 1.94 fold and 1.18 fold decreases, respectively, as compared to control plants. However, G1 and G2 plants showed a rise by 0.97 fold and 0.98 fold increase, respectively as compared to control plants. G1C, G2C and G3C plants showed 1.18 fold, twofold and 1.3 fold decline in shoot length when compared to control plants.
The fresh weight of the plant was seen to be affected by LT. Maximum decline of 2.1 fold fresh weight was reported in LT plants as that of control plants. G2  Stress tolerance index. Cold stress lowers the tolerance level of Pusa sheetal cv. of S. lycopersicum. However, exogenous supplementation of GSH tends to increase the tolerance level of the same. Root length stress tolerance index (RLSTI) and shoot length stress tolerance index (SLSTI) are maximally seen in GC2 plants (66% and 84% respectively) while least are seen in LT plants (40% and 53% respectively). The highest value of fresh weight (FW) and dry weight (DW) were recorded in GC2 plants (73% and 82.6%, respectively). The least value FW and DW was seen in LT plants (45.4% and 32% respectively). Marginal RLSTI (56%), SLSTI (79%), FWSTI (62%) and DWSTI (55.7%) was seen in G1C plants. G3C plants were noted to have RLSTI (50%), SLSTI (75%), FWSTI (61%) and DWSTI (63%) (Fig. 3).
Gas exchange parameters. There were significant variations in all of the gas exchange metrics examined across all treatments. Net photosynthetic rate (PN) was noticed remarkably lesser in the case of LT plants (7.88 µmolCO 2 m −2 s −1 ) as compared to control plants (11.56 µmolCO 2 m −2 s −1 ). GSH only treated plants G1, G2 and G3 plants have 11 Correlation based analysis 2D contour plotting. The 2D contour maps explain the response surface system. The oval shape of contour lines depicts the significant interaction between the variables while the insignificant type of interactions are generally shown by straight lines 22 . Here, in this analysis, the 3D form of data is visualized by a 2D pattern (Fig. 5). The distance between the contour lines represents the steepness of the slope, the lesser the distance, the more is the steepness (changing pattern of interaction), while the larger space between contour lines represents a soft slope (lesser changing pattern) while no lines show flat region (constant type of interaction   CAT activity. The activity of CAT enzyme in the leaves of S. lycopersicum seedlings was affected by LT stress and GSH treatment. LT + GSH (G1C, G2C and G3C) plants and only GSH (G1, G2, and G3) resulted in a surge in CAT activity. As compared to control plants 8.8 fold, 11.5 fold and 2.14 fold increase in CAT activity was deduced in G1, G2, and G3 treated plants, respectively. In comparison to the control plant, there is a little increase in CAT activity was also seen in LT only plants (Fig. 7a). However, in LT + GSH treated plants eightfold, 18.3 fold and 1.3 fold increase in CAT activity was seen in G1C, G2C and G3C, respectively. Hence, G2 concentrations have profoundly increased the activity of CAT under normal and LT stress conditions. Glutathione reductase activity. In the current study, the assessment of enzymatic activity of GR that is involved in preserving a high GSH/GSSG pool, which is critical for imparting tolerance under LT stress was done. Our study demonstrates, the level of GR activity in LT stressed and control plants was the same. However,  Superoxide dismutase activity. Evaluation of SOD enzyme activity that is seen as an initial line of defense to quench superoxide-free radicles generated due to LT stress was also measured. Plants treated with GSH had higher SOD activity than control plants, but LT plants had lower SOD activity. Only GSH treated plants G1, G2 and G3 showed 1.7 fold, twofold and 1.7 fold increase, respectively as that of control. LT + GSH treated plants including G1C, G2C and G3C have 1.8 fold, 2.14 fold, and 1.9 fold increase in SOD activity in comparison to that of LT stressed plants (Fig. 7c). SOD activity was better in G2C and least in LT plants. Thus, like other antioxidants SOD activity increased in presence of GSH.
Ascorbate peroxidase activity. LT (Fig. 7d). Henceforth, LT stress leads to a decline in APX activity while GSH application increases its activity in LT as well as in normal conditions.

Discussion
Under abiotic conditions, GSH is responsible for controlling a variety of physiological responses in plants but its role in mitigating cold stress tolerance in S. lycopersicum (Pusa sheetal cv.) has not yet been studied so far. In the present study, GSH was found to combat cold stress in S. lycopersicum by enhancing antioxidant machinery, biochemical traits, photosynthetic parameters and growth characteristics. LT stress has a significant effect on plant growth and developmental processes. Plant progress is influenced by LT. It initiates cascades of physiological, biochemical and morphological changes that limit the productivity of plants 23 . In the present study, LT caused a decrease in seedling growth with evident chilling injury as revealed by reduced SL, RL, FW and DW. It is in accordance with the previous as well as recent reports involving wheat 24 , barley 25 and rice 26 . There could be many reasons for the retarded growth of plants under LT stress like ROS production, improper nutrient uptake and osmotic imbalance 27 . However, GSH treated LT (GSH + LT) stressed seedlings were able to combat the detrimental effects of LT with better SL, RL, FW and DW (Fig. 8). Moreover, it is already reported that elevated levels of constitutive GSH increase cell division of the meristematic zone of the root region which leads to root elongation 28 . GSH treatment i:e; GSH only and LT + GSH enhanced root growth higher than control plants and LT-only plants, respectively. However, the progressive effect of exogenous GSH in controlling growth, www.nature.com/scientificreports/ development and yield under abiotic stress has been reported in Arabidopsis mung bean and soybean 11,29,30 . This study also indicates that GSH treatment has protective roles in lessening the toxic effects of LT on the growth and development of S. lycopersicum. It has been already reported that osmoregulators and antioxidants have a defined role in imparting abiotic stress tolerance like salinity stress 31 Moreover, the absorptive surface area of root (Fig. 8) were considerably elevated in GSH-supplied plants than in control and LT plants. This kind of scenario has also been described in maize under different abiotic stresses by Pei et al. 32 . Different root growth scenario was also evaluated in tomato cultivars under salt stress reported by Zaki et al. 33 . Photosynthesis is one of the principal physiological processes of plant systems that depend on various elements like light, fixation of CO 2 and other abiotic factors including temperature 34 . LT has been found to lower the CO 2 assimilation thereby reducing the rate of photosynthesis 35 . In the current experiment LT only plants have the least levels of photosynthesis as compared to the control. Exogenously applied GSH under normal or LT temperature tends to increase the rate of photosynthesis when compared to that of control and LT only respectively. This could be due to the thioredoxin property of GSH due which it controls the different enzymes of photosynthesis. However, it is also suggested that the reduced state of GSH in predominance protects the activity of the main enzymes of photosynthesis 36 . GSH probably protects the active sites from inhibitor binding that would halt the process of photosynthesis 37 . The transpiration rate, stomatal conductance and water usage efficiency increased in GSH treated plants as compared to LT only and the levels were comparable to that of control plants. This is in accordance with the photosynthetic performance shown by GSH increased transpiration rate (E), net photosynthetic rate (PN), and stomatal conductance (gs) abiotic stress in maize genotypes 38 . Furthermore, the membrane-stabilizing effect of GSH application could be a main protective mechanism for GSH-induced LT stress relief. GSH triggers signaling that regulates cellular redox state and protects fauna from abiotic stress while also maintaining cell membrane integrity 39,40 .
The ability of stomata to regulate their aperture to minimize water loss while maintaining CO 2 uptake is the intricate mechanism that favors the plant to persist under unfavorable conditions 41 . Stomatal movements (opening and closure) control the CO 2 usage and water loss via evaporation in response to environmental factors 42 . Decreased stomatal conductance results decline in the rate of photosynthesis by limiting the uptake of CO 2, while high stomatal conductance favors high photosynthesis output 43,44 . It is due to high stomatal conductance which causes the high rate of CO 2 uptake thereby increasing photosynthesis 42 . The cold stress causes the closure of stomata. Furthermore, it has been reported that abscisic acid (ABA) buildup occurs during LT stress in plants. The ABA has been reported to enhance stomatal closure. GSH depletion is also caused by ABA in guard cells. In addition, the GSH mutant plants show enhanced ABA-mediated stomatal closure 45 . But as per early reports, the GSH redox pool of cells has a promising effect on ABA signaling in plants 46 . The 2D contour maps are suggestive of significant interaction between variables while an insignificant type of interaction is shown by straight lines 22 . To demonstrate the type and the level of interaction between photosynthesis, the conductance of stomata, and transpiration rate 2D contour plots were taken into account. Contour plotting reveals that the interaction between these three parameters has an important role in combating cold stress tolerance in S. lycopersicum plants. From these plots, it is quite evident that G2C concentration of GSH favors this kind of interaction at maximum. Therefore, this kind of interaction seems to be an important factor that determines the LT stress alleviating capacity of the plants. More is the interaction between stomatal conductance, photosynthetic rate and transpiration rate more will be LT stress bearing tendency of S. lycopersicum plants. Our study also suggest that G2C favors this kind of interaction apart from boosting the antioxidant machinery of S. lycopersicum plants. This could be of suggestive that G2C concentration halts ABA mediated stomatal closure to much higher extend than G1C and G3C. As G2C concentration is almost having same levels or minute diminished levels of photosynthetic rate, stomatal conductance, and transpiration rate respectively in comparison with control. Meanwhile, G2C concentration helps to maintain its integrity by regulating stomatal conductance that which effect photosynthetic rate and transpiration rate. Hence, G2C may have promising role to enhance the GSH redox and inhibiting ABA mediating signaling in S. lycopersicum under cold stress conditions. The stress tolerance index (STI) is a valuable way of defining the stress tolerance potential. The SL, RL, FW and DW are important parameters for the classification of tolerance 47 . STI suggests a tolerance mechanism that allows plants to retain development even in the presence of abiotic factors such as hazardous metal levels 48 . In this study, maximum stress tolerance level was shown by G2C concentrations of GSH under LT stress followed by G1C and G3C. LT only plants decipher the least levels of STI. Consequently, GSH helps to increase STI of S. lycopersicum under LT stress. These findings correlate with data suggesting that the improved GSH level results in stress tolerance as seen in Arabidopsis 29 . In addition to imparting stress tolerance by GSH treatment, a rise in the components of the electron transport chain can be altered by GR activity. The involvement of GR is in preserving the decreased GSH level and control the cellular ROS scavenging phenomenon under stressful conditions 49 .
GSH being regarded as the main cellular antioxidant that acts as determining factor of the cellular redox state by regulating various redox signaling by reacting with ROS that is produced in response to various stresses thereby acting as a major scavenger 50,51 . Unfavorable environmental causes overproduction of ROS that leads to considerable changes in cellular lipid membrane causing peroxidation of lipids (shown by increased MDA) 52 . It was reported by Nahar et al. 13 that supplementation of GSH to mung bean seedlings improved tolerance level to high temperatures. Furthermore, Pei et al. 32 also reported that antioxidant activities involving SOD, CAT, GR and APX were reduced under abiotic treatment while the application of GSH enhanced their activity. Our results are in accordance with these findings that in imparting LT stress tolerance GSH is having important role via boosting the antioxidant capability of S. lycopersicum. Also, the level of membrane damage and H 2 O 2 content was seen least in plants treated with GSH. Hence, these results suggest that the decline in oxidative stress due to cold stress was ameliorated by GSH treatment which could be due to enhanced antioxidant capability thereby increasing stress tolerance observed in GSH-treated plants. Moreover, the reduced form of GSH directly detoxifies ROS and controls the activities pertaining to GSH dependent ROS and MG detoxifying enzymes 9 www.nature.com/scientificreports/ is an important part of the ascorbate -glutathione (AsA-GSH) cycle that regulates levels of H 2 O 2 in plant cells. Plants generally maintain high levels of GSH/GSSG ratio. GSH reacts with ROS species and gets transformed to GSSG causing a decline in GSH/GSSG ratio that results in oxidative stress 10,11 . So, the exogenous application of GSH keeps the GSH/GSSG pool in check to decrease the level of membrane damage apart from increasing antioxidant machinery. Among different GSH concentrations, G2 showed more promising antioxidant activities and lesser oxidative stress build-up in S. lycopersicum under LT stress. At higher concentrations, G3 response were not good as compared to G2 concentration. Reason could be that G2 concentration suit its physiological level as GSH treatment has been reported to increase the levels of ABA and jasmonic acid in plants. When plants being exposed to high concentration GSH causes evident rise in the endogenous level of ABA and Jasmonates that might have led to reduced growth and increase in LP even under normal conditions 29 .

Conclusion
In conclusion, exogenous supplementation alleviates the LT stress in Pusa sheetal cv. of S. lycopersicum plant. G2 concentration has immense potential to combat LT stress. Several means by which GSH encounters LT stress could be concluded in the following points.
(1) Maintaining osmotic equilibrium and membrane integrity. Our results provide insights on the role of GSH in combating LT stress and could be a possible approach to enhance LT stress resistance in Pusa sheetal cv. of S. lycopersicum.

Materials and methods
The Indian Agriculture Research Institute (IARI) in New Delhi, India, provided seeds of the Pusa Sheetal cv. of tomato. Surface sterilization of seeds with 2% sodium hypochlorite and then washed with sterile deionized water. In a growth chamber, these sterilized seeds were planted in a container containing soil made up of compost and peat (1/4, v/v) mixed with sand (3:1, v/v). 40 days after sowing (DAS), some plants were subjected to LT stress (10°/3 °C) day/night temperature (LT) for 24 h in a growth chamber rest kept at normal temperature 25/18 °C (control). While some plants were given folair GSH treatment of variable concentrations involving G1 (0.5 mM), G2 (1 mM) and G3 (2 mM) in prophylactic, preemptive and curative dosage dependent manner with LT (LT + GSH) and without LT (G). The LT stressed plants including LT + GSH and LT after 24 h were sustained under ambient conditions of day/night using 700 µmol m −2 s −1 photosynthetically active radiations, normal day/ night temperature of 25/17 ± 3 °C and relative humidity 75% in the growth chamber. Sampling was done after 3 days recovery period.
Growth data. Root length and shoot length. The root-shoot length specifies length of plant arise from the root tip to most growing tip of the central axis. Plants were uprooted carefully, washed and were retained on moist filter papers to avoid desiccation. With the aid of a measuring scale in cm, the root and shoot lengths were measured and recorded 54 .
Dry weight and fresh weight. Plants were uprooted cautiously, followed by proper washing to remove soil and weighed. Fresh weight will be deduced using balance. Plant dry mass was calculated after drying them at 80 °C in a hot air oven until constant weight is attained 55 . Determination of gas exchange parameters. Wholly expanded top most leaves of plants were analyzed under infrared gas analyzer (IRGA, Model LI6400XT, LI-COR Lincoln, Nebraska, USA) to determined Gas exchange parameters. The experiment was carried out between 11.00 and 12.00 h at light-saturating intensity, 2 cm 2 of leaf area, block temperature (25 °C), CO 2 flow controller (300 µmol s −1 ) and PAR (1600 μmol photons m −2 s −1 ). Before proceeding the experiment calibration of IRGA was done that includes zeroing replacement of drierite and soda lime. The healthy third leaf from apex was taken into account for recording leaf gaseous exchange attributes like transpiration rates (E) (mmol H 2 O m −2 s −1 ), stomatal conductance (gs) (mmol H 2 O m −2 s −1 ), photosynthetic rate (PN) (µmol CO 2 m −2 s −1 ) and water use efficiency (WUE). (Relationship between photosynthesis and transpiration).
Stress tolerance index. The tolerance indices for diverse growth factors were calculated using protocol followed by Amin H et al. 47 56 was followed. Samples were grounded in solution comprising of thiobarbituric acid (TBA 0.25%) prepared in trichloroacetic acid (TCA 10%) followed by heating them at 95 °C then cooled on ice and centrifuged for 10 min at 10,000 g. Subsequently 4 ml solution of TCA (20%) containing TBA (0.5%) was supplemented to 1 ml of supernatant. At 532 nm, the absorbance was measured. By subtracting the absorbance value of a comparable sample at 600 nm, the unspecific turbidity was adjusted. Using the extinction coefficient (155 mM -1 cm -1 ) the TBARS content was calculated.
The estimation of H 2 O 2 was determined suggested by Okuda et al. 57 . A fresh leaf sample was powdered in cooled perchloric acid (200 mM) for 10 min and then centrifuged at 1300 g. The supernatant containing perchloric acid was neutralized using 4 M potassium hydroxide (KOH). Centrifugation was used to remove the residual insoluble potassium perchlorate. The total amount of 1.5 ml contains eluate (1 ml), 3-methyl-2-benzothiazoline hydrazine (80 µl), 3-(dimethylamino) benzoic acid (12.5 mM, 400 µl) in phosphate buffer (0.375 M, pH 6.5) and 20 µl (0.25 unit) of peroxidase was prepared. At 590 nm, the increase in absorbance was recorded.
Catalase assay. The protocol of Aebi (1984) was used to deduce the CAT activity 58  Glutathione reductase assay. This activity was determined using Anderson 59 . The total reaction mixture (1 ml) have oxidized glutathione (0.02 mM, GSSG) and NADPH (0.2 mM) in a potassium phosphate buffer (0.1 M, pH 7.2). After adding enzyme extract (0.2 ml) to the mix, the process began. The activity was deciphered by fall in absorbance at 25 °C for 340 nm for 3 min. The conversion of 1 µmol of GSSG min -1 at 25 °C gives unit enzyme activity.
Superoxide dismutase. The Dhindsa et al. 56 method was followed to perform SOD assay relay on the capability of SOD to halt of formation of nitroblue tetrazolium (NBT) by photochemical reduction. The total reaction mixture consisting of 1.5 ml sodium phosphate buffer (0.1 M, pH 7.5) and PVP (1% w/v) L-methionine (13 mM), enzyme extract (0.1 ml) with same amounts of NBT solution (2.25 mM), riboflavin (60 μM), Na 2 CO 3 (1 M), EDTA (3 mM) and double-distilled water (1.0 ml, DDW). Samples were then irradiated at 28 °C under 15 W fluorescent lamp. At 560 nm the absorbance of the irradiated samples was compared to the non-irradiated samples. The quantity of enzyme extract equivalent to 50% reduction (Percent inhibition of colour) of NTB was taken as enzyme activity (single unit).
Ascorbate peroxidase activity (APX).. APX activity was carried out by the protocol of Nakano and Asada 60 . Centrifugation of Fresh leaf sample grounded in potassium-phosphate extraction buffer (0.1 M, pH 7, 5cm 3 ), Triton X 100 (1%), EDTA (3 mM), PVP (1%) was done at 4 °C for 10 min at 7800 g. The drop in ascorbate absorbance at 290 nm was used to calculate APX activity in the supernatant. Total reaction volume contains buffer (1 cm 3 ) contained ascorbate (0.5 mM), EDTA (0.1 mM), H 2 O 2 (0.1 mM) and extract of enzyme (0.05 cm 3 ). The reaction was proceeded at 25 °C for 5 min. By using coefficient of absorbance 2.8 mM -1 cm -1 APX activity was calculated. The amount essential to decompose 1 μmol of ascorbate per minute determines one unit of enzyme.

Statistical analysis.
Each experiment included the set three plants for each treatment. Graph-pad prism 8 software for Windows was used for statistical analysis. The analysis of variance (ANOVA) test was used to evaluate the significant differences between parameters. The value of p ≤ 0.05 was used to compare means. The data depicts the mean and standard deviation of three replicates. Data followed by ( * ) determines level of significance (p < 0.05) as predicted by Dunnet's multiple comparison test. 2D contour plots were plotted using Sigma 14. 5 Software package.
Ethical approval. The seeds utilized in this study were obtained from the Indian Agriculture Research Institute (IARI) in New Delhi, which governs the seed manufacturing and processing. This study complies with relevant institutional, national, and international guidelines and legislation.

Data availability
The datasets analyzed during the current study are not publicly available as it is meant to be published as Meta data sharing may publicise long term aim of this research however, data can be available from the corresponding author on reasonable request.